Distinct association of HRAS and KRAS with Mn2+ ion illustrated by paramagnetic NMR

Rat sarcoma virus oncogene (RAS) proteins are of crucial oncogenic proteins and are involved in several essential intracellular processes. The RAS protein has an intrinsic metal binding site for Mg²⁺, which is important for the conformational stability of the active site. Recently, it was reported that a second metal ion binding site, located further from the active site in HRAS (Harvey RAS homolog), binds Ca²⁺ with millimolar affinity. As one of the most abundant metal ions in cells, Mn²⁺ is a potential candidate for the second metal ion binding site in RAS proteins. Here, we examined the interaction of Mn²⁺ with HRAS and KRAS (Kirsten RAS homolog) using high resolution NMR spectroscopy. The NMR data showed that both the second metal ion binding site and the switch I and II regions bind Mn²⁺ in the RAS proteins. Furthermore, our paramagnetic NMR results disclosed the conformational differences in helix α3 and the following loop between HRAS and KRAS, accompanied by the association with metal ion binding. These results provide new insights into the interaction of RAS proteins and Mn²⁺ in the respective biological processes in cells.